Stem cell inhibitor

ABSTRACT

The present invention provides a Stem Cell Inhibitor (SCI) protein which comprises at least one amino acid alteration from its native form which protein does not significantly aggregate but which retains substantially unaltered stem cell inhibitory activity. The alteration is preferably a conservative subsitution of a charged amino acid residue. Such proteins may be used in treating stem cells in a patient undergoing chemotherapy.

This application is a divisional application of U.S. Ser. No.08/535,116, filed Jan. 29, 1996 and issued as U.S. Pat. No. 5,936,067;which was a national-phase application under 35 U.S.C. §371 fromPCT/GB94/00822 filed Apr. 19, 1994; which claimed the priority of UnitedKingdom application GB 9308060.4, filed Apr. 19, 1993; all of which areincorporated by reference herein.

The present invention relates to variants of stem cell inhibitors.

The treatment of cancer with chemotherapeutic agents is designed toattack and destroy cells which are undergoing division within the body.A side effect of such treatment is thus the destruction of normal cells,particularly the stem cells of the haematopoietic system and theepithelial stem cells which line the scalp and gut. Radiation can alsocause similar destruction of such cells.

It has been proposed that in order to improve the treatment of cancersby chemotherapy it would be desirable to protect stem cells from cellcycle specific cytotoxic drugs. WO89/10133 discloses a stem cellinhibitor and describes the use of the inhibitor in the treatment ofcancers. The inhibitor may be administered to a patient in order toprotect stem cells during chemotherapy.

Stem Cell Inhibitor (SCI), also known as MIP1-α is a peptide of about 8kD which forms large self aggregates, the molecular weight of which isdependant upon the concentration of SCI/MIP1-α monomers (Graham et al,1990, Nature 344;442, Wolpe & Cerami, 1989, FASEB J, 3; 2656). It hasbeen found that SCI/MIP1-α has a native, aggregated molecular weight ofabout 100 kD at 0.1 mg/ml in physiological buffers such as PBS. It hasbeen found that diluting SCI/MIP1-α to about 20-100 ng/ml or less willbring about disaggregation of this protein.

Human SCI/MIP1-α has been cloned by us (Graham et al (1992), GrowthFactors 7;151-160). The cDNA has also been cloned by Nakao et al (1990,Mol. Cell, Biol., 10;3646-58) and called LD78β. A variant of the cDNALD78α was also found, which has a very similar sequence. It differs byonly 4 amino acid residues. The human cDNA and protein sequence of thefactor cloned by us is shown is Seq. ID No. 1. The first 27 amino acidsare a leader sequence. The mature protein starts at residue 28 (ala).The amino acid sequence of the variant found by Nakao et al is shown asSeq. ID No. 3. The leader sequence of the protein is one amino acidshorter and thus the mature protein starts at residue 27 (ala). Thesequence of the murine homologue, upon which we have conducted our work,is also known and is very similar. It can be found for example in Grahamet al (1994, J. Biol. Chem., 269; 4974-78).

It has been reported (Mantel et al, 1993, PNAS 90;2232) that monomericSCI/MIP1-α is more active than the aggregated form in inhibiting invitro and in vivo stem cell proliferation. In using SCI/MIP1-α in thetreatment of humans it would be desirable to administer monomericprotein, not just from an activity point of view but also in order toprovide reliable and reproducible formulations. However, it is likelythat the low concentrations of SCI/MIP1-α which must be made in order toprovide monomeric protein will be too low for use in practice.

We have now surprisingly found that it is possible to obtain SCI/MIP1-αvariants which retain substantially the activity of the native proteinbut which do not form the same large aggregates. These mutants arestable as monomers or as small conglomerates (eg dimers or tetramers) atconcentrations many fold higher than native SCI/MIP1-α. Thus for thosevariants which have activity comparable to native SCI/MIP1-α, thevariants may have higher activity in vivo on a unit weight basis.

Accordingly, the present invention provides a Stem Cell Inhibitorprotein which comprises at least one amino acid alteration from itsnative form which does not significantly aggregate but which retainssubstantially unaltered stem cell inhibitory activity. The protein maycomprise either the full length stem cell inhibitor or the matureprocessed form lacking the leader sequence.

The invention also provides pharmaceutical compositions comprising astem cell inhibitor according to the invention in combination with apharmaceutically acceptable carrier or diluent, and optionally othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipients thereof.

The formulations include those suitable for parenteral (includingsubcutaneous, intramuscular, intravenous, intraperitoneal, intradermal,intrathecal and epidural) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. Such methods includethe step of bringing into association the active ingredient with thecarrier which constitutes one or more accessory ingredients. In generalthe formulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents, and liposomes or other microparticulate systems which aredesigned to target the compound to blood components or one or moreorgans. Suitable liquid carriers include phosphate buffered saline at apH of between 7.0 and 8.0, for example 7.4. The formulations may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of an activeingredient.

Formulations of the SCI/MIP-1α proteins of the present inventionpreferably contain from 0.05 to 5 mg/ml of protein, for example 0.1 to1.0 mg/ml. We have found that the solubility of the variants of theinvention do vary although the maximum solubility of any one particularvariant may be determined by simple titration by those of skill in theart.

The invention also provides such proteins and compositions for use in amethod of treatment of the human or animal body.

The invention further provides a method for treating a subject who is tobe exposed to an agent capable of killing dividing or cycling stem cellsby administering to the subject an effective amount of a protein orcomposition according to the invention.

The subject may also be treated with a protein or composition accordingto the invention during or after chemotherapy. In the latter case, thiswill usually be for a period sufficient to allow clearance of the agentfrom the body.

The method of treatment according to the invention may be used in thetreatment of solid tumours or leukemias. In the case of treatment ofleukemias, it is possible to treat a sample of the patients bone marrowwhich has been removed from the body while the patient is undergoingtreatment. The bone marrow is purged of cancer cells in the presence ofa protein of composition according to the invention, and the treatedmarrow reintroduced into the patient.

Although the dose of the variant protein according to the invention willultimately be at the discretion of the physician, taking into accountthe nature of the condition being treated and the state of the patient,effective doses may be in the range of from about 10 μg/kg body weightto about 5 mg/kg of variant protein, for example from about 50 to about1000 μg/kg, eg about 500 μg/kg.

We have also found that SCI/MIP1-α can act to enhance the expansion ofprimitive haemopoietic cells in ex vivo cytokine driven stem cellexpansion experiments. Thus, variant proteins of the invention may alsobe used in methods to expand stem cell populations removed from apatient ex vivo wherein such stem cells are brought into contact withgrowth factors and the variant proteins of the invention underconditions which allow the growth and expansion in numbers of the cells,prior to reintroduction into the same or another patient. Such a methodcould be used in bone marrow transplant proceedures whereby a limitednumber of starting cells obtained from a donor are expanded prior totransplantation, or in certain therapies where a sample of bone marrowis removed from a patient prior to treatment and reintroduced followingtreatment. Such therapies include the treatment of leukemias, or othertumours including solid tumours where damage to the bone marrow mayoccur. The concentration of the variant proteins required to producesuitable activity will be in the range of from about 1 to about 100ng/ml, for example from about 10 to about 50 ng/ml.

A protein or composition according to the invention may also be used inthe treatment of disorders caused by proliferation of stem cells, eg.psoriasis.

A protein according to the invention is preferably a protein whichcontains at least one change from the native protein resulting in theloss of of one of more charges on the protein, eg. by replacement of oneor more charged amino acids.

The change may be as a result of a deletion or substitution orinsertion. In the case of a deletion or insertion, single base deletionsor insertions are generally preferred, in order to retain a structuresimilar to the native protein. However, deletions of insertions of morethan this, eg or 2, 3, 4, 5 or more amino acids are possible. In thecase of a substitution, it is preferably a conservative substitution,such as Asp to Asn or Glu to Gln.

In addition, fragments of native protein which retain their stem cellinhibitory activity but which exhibit the reduced tendency to aggregateare within the scope or the invention.

Preferably, the change to the protein is in the C-terminal region, egwithin the last 20 or even last 10 amino acids. This may includeC-terminal deletions.

More than one change to a native stem cell inhibitor protein may bemade. For example, 2, 3, 4 or 5 changes may be made.

Another preferred region of the MIP1 protein which may be altered is theputative heparin binding region between amino acids 68 and 71 of Seq. IDNo. 1. We have determined by experimentation and by comparison of thissequence with known heparin binding regions that this portion of MIP1has heparin binding activity. Thus suitable amino acids which may bealtered in accordance with the invention include one, two or three of 68(lys), 69 (arg) and 71 (arg). Such alterations may be made, if desiredwith an alteration to the c-terminal region of the MIP1 protein asdescribed above.

Preferred stem cell inhibitor proteins of the invention are those basedupon the human protein of Seq. ID. 2 or that of Seq. ID 3. Alsopreferred are the mature forms of such proteins, ie. from residues 28onwards.

Particular amino acids which may be altered in the protein sequence ofSeq. ID No.2 or Seq. ID No. 3 include alterations at any positivelycharged residue, eg. lys or arg, and/or at any negatively chargedresidue, eg asp or glu. The residues of Seq. ID. No. 2 which may bealtered thus include: 29 (asp), 41 (arg), 50 (asp), 53 (glu), 60 (lys),68 (lys), 69 (arg), 71 (arg), 76 (asp), 79 (glu), 80 (glu), 84 (lys), 87(asp) or 90 (glu). The changes made to these positions may be asdescribed above.

Combinations of changes which may be made include changing the final 2,3, 4, 5 or 6 charged residues of the stem cell inhibitor. In the case ofthe human protein, this results in a protein which corresponds to thenative protein except for changes at position 90 and/or one or more ofpositions 76, 79, 80, 84 or 88. Preferably, all the changes are singleamino acid substitutions. Preferably, all such substitutions areconservative changes.

Proteins according to the invention may be made by any means availablein the art. In the examples which follow, we have made modified stemcell inhibitory proteins by site directed mutagenesis using PCR primersof the murine SCI cDNA, followed by expression of the modified cDNA in avector in a host cell to produce the protein. The protein may berecovered from the host cell using protein purification techniques knownper se. Analogous methods may be used to make modified human or otherprimate SCI. The murine cDNA may be obtained for example by reference tothe methods disclosed in WO89/10133 or by reference to the publishedliterature. Human cDNA may also be obtained by reference to thepublished literature or cloned using probes based on all or part of theDNA sequence of Seq. ID No. 1 to identify SCI cDNA in a cDNA librarymade from cells expressing SCI RNA.

Accordingly, the present invention also provides a method for making aprotein according to the invention which comprises:

(i) modifying a DNA sequence coding for SCI protein in order tointroduce at least one change which causes a change in the amino acidsequence of the SCI protein;

(ii) expressing said DNA, operably linked to a promoter, in a vector ina host cell compatible with said promoter; and

(iii) recovering said protein.

The DNA may be modified by site directed mutagenesis as mentioned aboveor described in the examples, to obtain insertions, deletions orsubsitutions in the amino acid sequence.

The vector may contain one or more selectable marker genes, for examplean ampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector.

A further embodiment of the invention provides host cells transformed ortransfected with the vectors for the replication and expression of DNAproduced as described above, including the DNA Seq. ID No. 1 modified asmentioned above. The cells will be chosen to be compatible with thevector and may for example be bacterial, yeast, insect or mammalian.

The invention also provides monoclonal or polyclonal antibodies to apeptide according to the invention which is directed to a epitopecontaining an alteration of the native SCI. The invention furtherprovides a process for the production of such monoclonal or polyclonalantibodies. Monoclonal antibodies may be prepared by conventionalhybridoma technology using the proteins or peptide fragments thereof, asan immunogen. Polyclonal antibodies may also be prepared by conventionalmeans which comprise inoculating a host animal, for example a rat or arabbit, with a peptide of the invention and recovering immune serum.

In either case, antibodies which recognise altered epitopes may beidentified by screening them with native SCI and the altered SCI towhich the antibody was raised and identifying an antibody whichrecognises only the altered SCI.

The following examples illustrate the invention.

EXAMPLE 1

FIG. 1 shows a schematic representation of murine SCI/MIP1-α indicatingthe position of charged amino acids. A series of altered proteins(1)-(3) were made using PCR primers on cDNA encoding the proteintogether with a wild type 5′ primer. The altered proteins all containedconservative changes, ie. glutanmate to glutamine and/or aspartate toasparagine. The primers used are as follows:

Variant 1

5′ TC AGG AAT TCA GGC ATT CAG TTG CAG GTC 3′ (SEQ ID NO. 4). This altersthe C-terminal end of the murine MIP1-α protein from: VQEYITDLELNA (SEQID NO. 5) to VQEYITDLQLNA (SEQ ID NO.6).

Variant 2

5′ TC AGG AAT TCA GGC ATT CAG TTG CAG GTT AGT GAT 3′ (SEQ ID NO.7) whichalters Seq. ID No. 5 to VQEYITNLQLNA (SEQ ID NO.8).

Variant 3

5′ TC AGG AAT TCA GGC ATT CAG TTG CAG GTT AGT GAT GTA TTG TTG GAC 3′(SEQ ID NO. 9) which alters Seq. ID No. 5 to VQQYITNLQLNA (SEQ ID NO.10)

The varied cDNA molecules were ligated into a fusion protein expressionvector and the altered proteins were produced. The native proteintogether with the three altered proteins were analysed bychromatographic techniques and the molecular weights of each estimated.

The estimates were as follows:

Native protein 100-150 kD Protein (1) 35 kD Protein (2) 18 kD Protein(3) 8 kD

Protein (1) thus appears to exist as a tetramer, protein (2) as a dimerand protein (3) as a monomer under conditions in which native MIP1-αexists as an aggregated protein.

The above proteins were assesed for bioactivity using standardtechniques (Pragnell et al Blood, 1988, 72; 196 and Lorimore et al,1990, Leukaemia Research 14; 481) and found to be bioactive.

EXAMPLE 2

Two 3′ (carboxy terminus) primers were synthesised with the followingsequences:

5′ GTA CGT GGA TCC TCA GGC ACT CAG CTG CAG GTT GCT GAC ATA TTG CTG GAC3′ (SEQ ID NO. 11) and

5′ GTA CGT GGA TCC TCA GGC ACT CAG CTG CAG GTT GCT GAC ATA TTG CTG GACCCA CTG CTC ACT 3′ (SEQ ID NO. 12).

A Bam H1 recognition site is underlined.

The primer of Seq. ID No. 11 encodes amino acids 82 to 93 of Seq. ID No.1 but alters the lysine at position 84 (84 (lys)) to glutamine (gln), 88(asp) to asn, and 90 (glu) to gln.

The primer of Seq. ID. No. 12 encodes to amino acids 78 to 93 of Seq. IDNo. 1 but contains the three changes described above for Seq. ID No. 11and also a further change, 80 (glu) to gln.

To produce the human variants incorporating the above changes the aboveprimers are each used with an amino terminal primer of Seq. ID No. 13:

5′ GAC GGC CAT GGC TGA CAC GCC GAC CGC CTG C 3′ (SEQ ID NO. 13) whichencodes amino acids 28-35 of Seq. ID No. 1. An Nco1 recognition site isunderlined. This corresponds to the start of the mature SCI/MIP-1protein.

The primers are used in a PCR to provide full length clones encodingvariants incorporating the changes described above, and the variantclones introduced into an expression vector to provide dissagregatedvariant proteins of the invention.

The variants are tested in a similar manner as described above foractivity.

EXAMPLE 3

A internal primer which encodes a central portion of the murine MIP1-αprotein was designed, incorporating changes which cause point mutationsin two of the three positively charged residues between the third andfourth cysteine residues shown in FIG. 1. The primer is of the sequence:

5′ CGT CTA GAC GGC CAA CGA CAA TCA GTC CTT 3′ (SEQ ID NO. 14) whichalters the murine sequence:

FLTKRNRQIC (SEQ ID NO. 15) to FLTNSNRQIC (SEQ ID NO. 16).

The mutagenesis was done in two halves using this primer and the wildtype amino terminal primer and a complemetary primer was used with thewild type carboxy terminal primer. The two reaction products were thenmixed and the full length molecule produced using the wild type aminoand carboxy terminal primers. The variant is also tested for activity.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 16(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 282 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide     (ix) FEATURE:          (A) NAME/KEY: CDS           (B) LOCATION: 1..282    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:ATG CAG GTC TCC ACT GCT GCC CTT GCC GTC CT#C CTC TGC ACC ATG GCT       48Met Gln Val Ser Thr Ala Ala Leu Ala Val Le #u Leu Cys Thr Met Ala  1               5  #                 10  #                 15CTC TGC AAC CAG GTC CTC TCT GCA CCA CTT GC#T GCT GAC ACG CCG ACC       96Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Al #a Ala Asp Thr Pro Thr             20      #             25      #             30GCC TGC TGC TTC AGC TAC ACC TCC CGA CAG AT#T CCA CAG AAT TTC ATA      144Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Il #e Pro Gln Asn Phe Ile         35          #         40          #         45GCT GAC TAC TTT GAG ACG AGC AGC CAG TGC TC#C AAG CCC AGT GTC ATC      192Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Se #r Lys Pro Ser Val Ile     50              #     55              #     60TTC CTA ACC AAG AGA GGC CGG CAG GTC TGT GC#T GAC CCC AGT GAG GAG      240Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Al #a Asp Pro Ser Glu Glu 65                  # 70                  # 75                  # 80TGG GTC CAG AAA TAC GTC AGT GAC CTG GAG CT #G AGT GCC TGA             # 282 Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Le #u Ser Ala  *                 85  #                 90(2) INFORMATION FOR SEQ ID NO:2:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 93 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:Met Gln Val Ser Thr Ala Ala Leu Ala Val Le #u Leu Cys Thr Met Ala  1               5  #                 10  #                 15Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Al #a Ala Asp Thr Pro Thr             20      #             25      #             30Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Il #e Pro Gln Asn Phe Ile         35          #         40          #         45Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Se #r Lys Pro Ser Val Ile     50              #     55              #     60Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Al #a Asp Pro Ser Glu Glu 65                  # 70                  # 75                  # 80Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Le #u Ser Ala                 85  #                 90(2) INFORMATION FOR SEQ ID NO:3:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 92 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:Met Gln Val Ser Thr Ala Ala Leu Ala Val Le #u Leu Cys Thr Met Ala1               5    #                10   #                15Leu Cys Asn Gln Phe Ser Ala Ser Leu Ala Al #a Asp Thr Pro Thr Ala            20       #            25       #            30Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pr #o Gln Asn Phe Ile Ala        35           #        40           #        45Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Ly #s Pro Gly Val Ile Phe    50               #    55               #    60Leu Thr Lys Arg Ser Arg Gln Val Cys Ala As #p Pro Ser Glu Glu Trp65                   #70                   #75                   #80Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Se #r Ala                 85  #                90 (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 29 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:TCAGGAATTC  AGGCATTCAG  TTGCAGGTC        #                  #            29 (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 12 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:Val Gln Glu Tyr Ile Thr Asp Leu Glu Leu As #n Ala 1               5   #                10 (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 12 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:Val Gln Glu Tyr Ile Thr Asp Leu Gln Leu As #n Ala 1               5   #                10 (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 35 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:TCAGGAATTC AGGCATTCAG TTGCAGGTTA GTGAT        #                  #       35 (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 12 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:Val Gln Glu Tyr Ile Thr Asn Leu Gln Leu As #n Gln 1               5   #                10 (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 47 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:TCAGGAATTC  AGGCATTCAG  TTGCAGGTTA GTGATGTATT  GTTG#GAC                47 (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 12 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:Val Gln Gln Tyr Ile Thr Asn Leu Gln Leu As #n Ala 1               5   #                10 (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 51 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:GTACGTGGAT CCTCAGGCAC TCAGCTGCAG GTTGCTGACA TATTGCTGGA C #             51 (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 63 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:GTACGTGGAT CCTCAGGCAC TCAGCTGCAG GTTGCTGACA TATTGCTGGA CC#CACTGCTC     60 ACT                   #                  #                   #             63 (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:GACGGCCATG GCTGACACGC CGACCGCCTG C         #                  #          31 (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:CGTCTAGACG  GCCAACGAC AATCAGTCCTT         #                  #           30 (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:Phe Leu Thr Lys Arg Asn Arg Gln Ile Cys 1               5   #                10 (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:Phe Leu Thr Asn Ser Asn Arg Gln Ile Cys 1               5   #                10

What is claimed is:
 1. A protein having stem cell inhibitory activityand having an amino acid sequence comprising the sequence of SEQ ID NO:2from amino acid 1 through amino acid 93 with at least one amino acidsubstitution at an amino acid selected from the group consisting of29(asp), 41(arg), 50(asp), 53(glu), 60(lys), 68(lys), 69(arg), 71(arg),76(asp), 79(glu), 80(glu), 84(lys), 88(asp) and 90(glu).
 2. A proteinaccoding to claim 1 wherein the amino acid substitution results in theloss of a charged amino acid.
 3. A protein according to claim 1 whereinthe substitution is a conservative substitution.
 4. A protein accordingto claim 3 wherein the substitution is of Asp to Asn, or of Glu to Gln.5. A protein according to claim 1 which contains 2 or 3 amino acidsubstitutions.
 6. A pharmaceutical composition comprising a proteinaccording to claim 1 in combination with a carrier or diluent.
 7. Amethod for treating a subject who is to be exposed to an agent capableof killing dividing or cycling stem cells by administering to thesubject an amount of a composition according to claim 6 effective toinhibit the proliferation of hematopoietic stem cells wherein saidtreatment is protection of hematopoietic stem cells from killing by saidagent.
 8. A method for making the protein of claim 1 which comprises:(i) expressing a polynucleotide encoding the protein of claim 1 operablylinked to at least one promoter, in a vector in a host cell compatiblewith said promoter; and (ii) recovering said protein.